RERF scientific agenda and DOE.
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Foundations↗
Biomedical subjects
C Satoh
Publications and source records attributed to C Satoh.
Radiation studies.
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Government Agencies↗
High yield of restriction fragment length polymorphisms in two-dimensional separations of human genomic DNA.
We have investigated the extent to which restriction fragment length polymorphism can be detected by two-dimensional electrophoresis of end-labeled genomic restriction fragments. Genomic DNA was digested with NotI and EcoRV and labeled at the NotI recognition site before first-dimension electrophoresis in disk gels. DNA in the disk gels was further digested in situ with HinfI prior to second-dimension electrophoresis, yielding patterns in which approximately 2000 end-labeled fragments were simultaneously visualized. On the basis of studies of 6 mother/father/child trios, a group of 184 fragments was organized into 85 polymorphic systems in which all allelic fragments were detectable in the 2-D patterns. Another 206 fragments varied as to their presence among individuals, but their relatedness to other fragments was not established. Our data indicate that a large number of DNA polymorphisms can be simultaneously scored in 2-D separations of genomic DNA fragments.
Adult↗
Quantitative and qualitative genetic variation in two-dimensional DNA gels of human lymphocytoid cell lines.
There is a continuing need for more efficient methods to examine human (and other) populations for altered germinal and somatic cell mutation rates. To this end, we have explored the potential usefulness of two-dimensional (2-D) electrophoresis of human DNA fragments obtained from restriction-enzyme-digested genomic DNA, using samples from father/mother/child trios. On a single 2-D DNA preparation, approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kbp in the first dimension and 0.3 to 2.0 kbp in the second dimension are visualized. To enter into a genetic analysis of quantitative variation, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately < or = 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variations that segregated within families according to Mendelian principles. Additionally, of the 2000 spots, 1114 (of which the aforementioned 482 are a subset) were deemed appropriate for the study of qualitative variation. A total of 142 variable spots were identified; the heterozygosity index for these DNA fragments was 4.4%. The genetic nature of the additional variants was again established by their segregation according to Mendelian principles. We have established the feasibility of cloning fragments from such gels and determining their nucleotide sequence. This technology should be highly efficient in monitoring for mutation resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.
Autoradiography↗
Genetic variation detected by quantitative analysis of end-labeled genomic DNA fragments.
The continuing efforts to evaluate specific human populations for altered germinal mutation rates would profit from more efficient and more specific approaches than those of the past. To this end, we have explored the potential usefulness of two-dimensional electrophoresis of DNA fragments obtained from restriction-enzyme-digested genomic DNA. This permits the analysis, on a single preparation, of approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kb in the first dimension and from 0.3 to 2.0 kb in the second dimension. To enter into a genetic analysis, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately < or = 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variation that segregated within families according to Mendelian principles. We have established the feasibility of cloning a variant fragment from such gels and establishing its nucleotide sequence. This technology should be highly efficient in monitoring for mutations resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.
Base Sequence↗
Decreased E-cadherin expression in endometrial carcinoma is associated with tumor dedifferentiation and deep myometrial invasion.
Decreased E-cadherin expression in tumor cells has been suggested to promote tumor invasiveness. We examined E-cadherin expression in 30 cases of endometrial carcinoma by immunohistochemistry using a monoclonal antibody to E-cadherin and investigated its correlation with other histopathologic features of the tumor. We observed that: (1) E-cadherin expression decreased with loss of differentiation (P < 0.05); (2) E-cadherin expression was inversely correlated with depth of myometrial invasion (P < 0.05); (3) decreased E-cadherin expression was correlated with paraaortic node metastasis (P < 0.01); and (4) multivariate analysis comparing the depth of myometrial invasion to the pattern of E-cadherin expression, histologic grade, nuclear grade, and lymph-vascular space invasion showed that the depth of myometrial invasion was most strongly correlated with decreased E-cadherin expression (P < 0.005). These findings seem to be consistent with the concept that the dissociation of cancer cells due to decreased expression of E-cadherin facilitates invasion of tumor cells.
Analysis of Variance↗
[A clinical and pathological study on paraaortic lymph node metastasis in uterine cervical carcinoma].
Three hundred and three patients with uterine cervical cancer underwent radical hysterectomy including Paraaortic lymph node (PAN) dissection between June, 1982 and March, 1990. We analyzed the relationship between PAN metastasis and the clinical stage, histologic type, parametrial invasion and pelvic lymph node (PLN) metastasis, and obtained the following results. 1) The incidence of PAN metastasis in clinical stages Ib, II and III was 0.9%, 4.5% and 16.7%, respectively. 2) The PAN metastasis rate in case of adenocarcinoma was 8.3% which tended to be higher than that for squamous cell carcinoma (4.9%) and mixed carcinoma (3.2%). 3) Deeply invading carcinoma penetrating to the parametrium (including the parametrial initial zone) was seen in 15 out of 16 cases (93.8%) with PAN metastasis. PAN metastasis was rare in cases without parameterial invasion (1/114). 4) Positive PAN was found in 48.3%, 34.8% and 41.9% of cases with metastasis in more than four lymph node groups, bilateral PLN and common iliac lymph node (CILN), respectively. The present results suggest that a regular careful check up for PAN metastasis is necessary for case with multiple PLN metastasis (bilateral and more than four groups) and CILN metastasis.
Adult↗
Duplication detection in Japanese Duchenne muscular dystrophy patients and identification of carriers with partial gene deletions using pulsed-field gel electrophoresis.
DNA samples from 21 unrelated Japanese patients with Duchenne muscular dystrophy (DMD) with nondeletion-type abnormality in the dystrophin gene and three samples from possible deletion carriers were analyzed using pulsed-field gel electrophoresis (PFGE). Among the 21 patients, 7 were found to carry partial duplications of the dystrophin gene spanning 50-400 kb. Of these 7 patients, 4 carried duplications corresponding to the major hot-spot regions for deletions (7.5-8.5 kb from the 5' end of cDNA), whereas two cases contained duplications in a region about 10 kb from the 5' end of cDNA, where causative mutations are reported to be rare. Only 1 case was found to contain a duplication of a region about 1 kb from the 5' end of cDNA, which is the reported duplication prone region. A combination of Southern blot analyses of conventional agarose gel electrophoresis and PFGE was confirmed to be useful, not only for detecting duplications and deletions, per se, but also for identifying carriers in the affected family.
Blotting, Southern↗
Detection of partial deletion and partial duplication of dystrophin gene in Japanese patients with Duchenne or Becker muscular dystrophy.
The dystrophin gene was analyzed in 59 Japanese patients with Duchenne muscular dystrophy (DMD) from 48 unrelated families, including 11 pairs of siblings, and three patients with Becker muscular dystrophy (BMD) from two unrelated families, including one pair of siblings. The relationship between the type of gene abnormality and clinical symptoms was examined. Twenty-seven of 50 (54.0%) unrelated DMD or BMD patients were found to have partial deletions, and five (10%) appeared to have partial duplications in the dystrophin gene. Nine DMD patients, including three pairs of siblings, showed mental retardation, the existence of which was coincident in each pair of siblings, but deletion of an identical exon was not always related to mental retardation in unrelated patients.
Adolescent↗
International Commission for Protection against Environmental Mutagens and Carcinogens. Report of a workshop on the application of molecular genetics to the study of mutation in the children of atomic bomb survivors.
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Child↗
[Accurate detection of heterozygous carriers of a deletion or a duplication by combined PCR and HPLC].
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Chromatography, High Pressure Liquid↗
Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis.
In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE--a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies.
Base Sequence↗
Accurate and rapid detection of heterozygous carriers of a deletion by combined polymerase chain reaction and high-performance liquid chromatography.
We have developed a technique to detect accurately heterozygous carriers of a deletion. Specific target sequences were amplified by the polymerase chain reaction (PCR), and the products subsequently were analyzed by high-performance liquid chromatography. Examples from four loci demonstrated that 24-27 cycles of amplification for a single-copy DNA, based on 50 ng of genomic DNA, results in excellent quantitation that readily permits the detection of heterozygous carriers of a deletion. We have demonstrated that triplex PCR (three targets in a single PCR) entails no loss of precision. We also have demonstrated that this method can accurately differentiate the heterozygous carriers of a deletion from normal individuals in four family studies, three for Duchenne muscular dystrophy patients and one for a hemophilia B patient.
Base Sequence↗
[Effects of self-efficacy and outcome expectation on observational learning of altruistic behavior].
The purpose of the present study was to examined the hypothesis that the effects of observational learning of altruistic behavior are related to personal viewpoint, self-efficacy, outcome expectation, and other factors. One hundred and fifty-five fourth-grade school children were assigned randomly to four conditions; distress viewpoint, outcome expectation, altruistic behavior, or non-observational control. After observational learning, the students were immediately administered the generalization test of altruistic behavior, empathy, reward and punishment expectations, and rating tests of self-efficacy belief. It was found that; (a) altruistic behavior and outcome expectation conditions had significant learning effects, (b) self-efficacy was able to predict altruistic response in the altruistic behavior viewpoint condition, and (c) from an outcome expectation viewpoint, altruistic behavior toward peers and adults related to punishment expectation, whereas social sharing was related to reward expectation. According to these findings, an observational learning model of altruistic behavior was proposed.
Altruism↗
[An analysis on prognostic significance of histopathologic risk factor in uterine endometrial carcinoma].
Prognostic risk factors in histopathologic findings were analyzed in the data for one hundred and thirteen patients with uterine endometrial carcinoma who were treated surgically. Univariate survival analysis with Kaplan-Meier methods revealed that the nuclear grade (p less than 0.005), lymph-vascular space invasion (p less than 0.005), histologic grade (p less than 0.01) and histologic type (p less than 0.01) correlated with the patient's prognosis. Among surgical FIGO stages II and III, there was similar significance in the correlations in the nuclear grade (p less than 0.05), lymph-vascular space invasion (p less than 0.0001) and histologic type (p less than 0.05), although there was difference (p less than 0.05) only between grade 1 and grade 3 in the histologic grade. Multivariate survival analysis with a proportional hazard regression model showed that the nuclear grade (p less than 0.005) and lymph-vascular space invasion (p less than 0.01) correlated significantly with the prognosis. The hazard ratios with a 95% confidence interval for each of these factors were 19.2 (3.2-115.7) and 16.9 (2.1-135.3), respectively. The 3-year survival rate was 98% for a hazard ratio less than 64.7, and 45% for one more than 67.4, between which there was a significant difference (p less than 0.0001). And univariate survival analysis of this hazard ratio also revealed strong correlations with the patient's prognosis. These results suggest that it is of great importance to adopt the hazard model including such factors as the nuclear grade and lymph-vascular space invasion in estimating the patient's prognosis and proceeding to adequate post-operative therapy for individual patient.
Adenocarcinoma↗
Re-transformation of non-transformed hybrids between c-myc-activating mouse plasmacytoma cells and normal fibroblasts by transfection with activated c-Ha-ras but not c-myc.
In a mouse plasmacytoma S194, c-myc oncogene is rearranged with Ig gene by chromosomal translocation and is consequently activated. We previously reported that transformation of phenotype and expression of rearranged c-myc were repressed in independently isolated hybrid clones, I-1 and IV-10, between S194 and normal fibroblasts. In order to investigate the relationship between transformation of phenotype and oncogene expression, transcriptionally enhanced c-myc or activated c-Ha-ras was transfected into I-1 or IV-10I, a subclone of IV-10. Transfectants expressing high levels of c-myc were found to retain the non-transformed phenotypes. On the other hand, transfectants expressing activated c-Ha-ras showed the transformed phenotypes. These results suggest that enhanced expression of c-myc is not sufficient for re-transformation of the non-transformed hybrid clones between c-myc-activating plasmacytoma cells and normal fibroblasts, but expression of activated c-Ha-ras could diminish or overcome the tumor-suppressive activity of normal fibroblasts.
Animals↗
Chromosome translocation and c-MYC activation by Epstein-Barr virus and Euphorbia tirucalli in B lymphocytes.
Dual exposure to Epstein-Barr virus and purified 4-deoxyphorbol ester derived from the plant Euphorbia tirucalli induced a high frequency of chromosomal rearrangements in human B lymphocytes in vitro. Rearrangements most commonly affected chromosome 8, the chromosome most often showing structural changes in Burkitt's lymphoma (BL) cells. E tirucalli is indigenous in parts of Africa where BL is endemic and may be an important risk factor for the disease.
B-Lymphocytes↗
The length polymorphism in the 5' flanking region of the human beta-globin gene with denaturing gradient gel electrophoresis in a Japanese population.
The ATTTT repeat polymorphism located approximately 1,400 base pairs (bp) upstream from the beta-globin structural gene was analyzed by denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes. A study of 81 unrelated Japanese from Hiroshima revealed a sequence heteromorphism in this site. The alleles with five and six repeats of the ATTTT unit, which have been reported, were found in polymorphic proportions. Two unreported alleles were also detected, the first, in two persons, characterized by seven repeats and the other, in a single person, having an A-to-G nucleotide substitution in the fifth repeat.
Cell Line↗